Vesicle including a metal marker for use in an assay

ABSTRACT

Vesicles are formed, in part, from an amphiphilic chelating agent whereby a detectable metal marker can be complexed to the vesicle. A portion of the vesicle may also be formed from an amphiphilic compound derivatized with a ligand, whereby the vesicle may be used as a tracer in an assay. If the detectable metal marker is a fluorescent rare earth metal, the assay may be a time delay fluorescent assay. The vesicles may be dried, and reformed by addition of water for use in an assay.

This invention relates to sacs including a detectable marker, and theuse thereof in an assay for an analyte. This invention further relatesto saces including a detectable marker which are sensitized with aligand and the use thereof in an assay for an analyte.

Sacs, and in particular, lipid vesicles, have been prepared which have adetectable marker encapsulated therein. Such sacs have been employed inassays for a ligand (analyte). In a representative assay, a ligand to bedetermined (analyte) and tracer comprised of a sac having a detectablemarker encapsulated therein, which sac is also sensitized with theanalyte or appropriate analog thereof, compete for a limited number ofbinding sites on a binder for the analyte. The amount of tracer whichbecomes bound to the binder is inversely proportional to the amount ofanalyte in the sample. After separating bound and free tracercomponents, the amount of the bound and/or free tracer is ascertained bydetermining the detectable marker in the bound and/or free tracerportion of the sample, which provides a measure of analyte in thesample.

The assay provides for increased sensitivity, and amplification ofsignal in that a single ligand molecule, which is employed in formingthe tracer, includes a plurality of marker components.

In many cases, however, the sacs having the detectable markerencapsulated therein do not possess sufficient stability in that thedetectable marker "leaks" from the sacs prior to or during the assay,which limits the effectiveness of such sacs in an assay. The term"leaks", as used herein, means that either the material escapes from anintact sac or the material escapes as a result of destruction of thesac.

In addition, the sac having the marker encapsulated therein must bemaintained in water so as to maintain the sac structure. This increasesthe storage problems associated with sacs including an encapsulatedmarker.

In accordance with one aspect of the present invention, there isprovided a particle which includes both a detectable metal marker and aligand, which is particularly suited for use in an assay.

In accordance with another aspect of the present invention, there isprovided a vesicle in which a portion of the vesicle is formed from anamphiphilic chelating agent.

In accordance with yet another aspect of the present invention, thereare provided amphiphilic chelating agents which are particularly suitedfor use in forming vesicles.

In accordance with a further aspect of the present invention, there isprovided a vesicle in which a portion of the vesicle wall is formed froman amphiphilic chelating agent having a detectable metal marker complexthereto, and another portion of the wall is formed from an amphiphiliccompound sensitized with a ligand.

In accordance with yet a further aspect of the present invention, thereis provided an assay which employs vesicles and particles of the typehereinabove described.

In accordance with still another aspect of the present invention, thereis provided a hapten labeled with a plurality of detectable metal atoms.

More particularly, particles are provided with a chelating agent forcomplexing a detectable metal, and in accordance with a preferredaspect, such particles are vesicles, with a portion of the vesicle beingformed from an amphiphilic chelating agent.

Vesicles may be prepared from one or more amphiphilic compounds(compounds including both a hydrophilic portion or moiety and ahydrophobic portion or moiety, which are capable of forming a vesiclewall). In most cases, at least one of the components used in preparingthe vesicle is a lipid, and such vesicles are commonly referred to asliposomes, even though the vesicles may be prepared from amphiphiliccompounds other than lipids.

In accordance with an aspect of the present invention, a portion of thevesicle is formed from an amphiphilic chelating agent which is comprisedof a hydrophilic chelating portion, and a hydrophobic portion. Suchamphiphilic chelating agents may be prepared by conjugating orderivatizing a chelating agent with a compound including a hydrophobicmoiety of the type generally employed in producing vesicles. Thus, forexample, a chelating agent may be derivatized with or conjugated to aphospholipid, such as a phosphatidylethanolamine; a steroid, such ascholesterol; a glycolipid; a long chain dialkylamine; a long chaindialkylcarboxylic acid or ether; an ester of a polyhydroxyalcohol, suchas glycerol, so as to provide an amphiphilic chelating agent which isemployed to form a portion of the sac.

Any one of a wide variety of chelating agents may be employed for suchpurpose, and as representative examples of such chelating agents, theremay be mentioned aminocarboxylic acids, iminocarboxylic acids, ethers,thiols, phenols, glycols and alcohols or polyamines,ethylenediaminetetracetates, diethylenetriaminepenta or tetracetates,polyethers, polythiols, cryptands, polyetherphenolates, polyetherthiols, ethers of thioglycols or alcohols, polyaminephenols, all eitheracyclic, macrocyclic, cyclic, macrobicyclic or polycyclic, or othersimilar ligands which produce highly stable metal chelates or cryptates.

The selection of a particular chelating agent will depend upon the metalto be chelated, as well as the wall forming material to which thechelating agent is to be conjugated.

The chelating agent is conjugated to a compound including a hydrophobicmoiety to provide an amphiphilic chelating agent through a suitablefunctional group. As representative examples of suitable functionalgroups, there may be mentioned: carboxylic acid groups, diazotiazableamine groups, succinimide esters, anhydrides, mixed anhydrides,benzimidates, nitrenes, isothiocyanates, azides, sulfonamides,bromoacetamides, iodoacetamides, carbodimides, sulfonylchlorides,hydrazides and thioglycols. Such functional groups may be included inthe hydrophobic moiety and/or chelating agent to permit direct linkageor the hydrophilic chelating agent may be conjugated to the hydrophobicmoiety through a suitable spacer group.

Thus, for example, suitable spacer compounds include diamines, such ashexamethylenediamine; diaminocarboxylic acids, such as lysine;polyethers; polyalcohols; aminoalcohols; aminopolycarboxylic acids,polycarboxylic acids; polyhydroxycarboxylic acids; hydroxypolycarboxylicacids, etc.

The selection of suitable spacer compounds and functional groups forconjugating a chelating agent to a compound including a hydrophobicmoiety to provide an amphiphilic chelating agent suitable for use inproducing a vesicle is deemed to be within the scope of those skilled inthe art from the teachings herein. A particular preferred type oflinkage is an amide linkage formed by conjugating a carboxyl group of achelating agent to an amino group of a compound including a hydrophobicmoiety. For example, such amide linkages may be formed by conventionalpeptide synthesis methods.

The chelating agents which are particularly preferred areaminocarboxylic acids; and in particular, ethylenediaminetetraaceticacid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). Suchchelating agents may be conjugated or derivatized; for example, with along chain dialkylamine; or with a phosphatidylethanolamine or with asteroid, such as cholesterol or cholestanol to provide an amphiphilicchelating agent suitable for use in forming a vesicle.

As representative examples of preferred amphiphilic chelating agents,there may be mentioned those represented by the following structuralformulas: ##STR1## wherein each R is an alkylene group orp-diazon-iumphenyl substituted alkylene group wherein the alkylene grouphas from 2-4 carbon atoms and each R group may be the same or different,

X and Y are integers from 0-8,

A is hydrogen, or W;

one W group is ##STR2## or B--(PO₄)--(CH₂)₂ --NH--C(0)--CH₂ ; or A₁ --B₂--C(0)--CH₂ --

wherein R' is an alkylene group having from 2-10 carbon atoms; R" is analiphatic hydrocarbon having at least 11 carbon atoms and may be thesame or different and Z is an integer of from 0-4, and the remaining Wgroups are --CH₂ --C(0)--OR"'

wherein R"' is hydrogen, an alkali metal or an amine, and B is ##STR3##wherein Y is an aliphatic hydrocarbon radical having from 1-5 carbonatoms, preferably 3 or 4 carbon atoms; each of B₁ is individually --0--;--C(0)0--;

or --CH₂ -- and may be the same or different; and each R₁ is asubstituted or unsubstituted hydrocarbon radical (saturated orunsaturated) having at least 11 carbon atoms and may be the same ordifferent,

and A₁ is cholesterol or cholestanol and B₂ is a radical formed from adifunctional spacer compound.

Particularly preferred compounds of this type are a dialkylamineconjugated to ethylenediaminetetraacetic acid or salt thereof (X and Zare zero, R is ethylene); and a dialkylamine conjugated todiethyleneaminepentaacetic acid or salt thereof (R is ethylene, Y is 1,X and Z are zero).

Another preferred compound is one in which W is A₁ --B₂ --C(0)--CH₂ ⁻,

A₁ is a cholesterol radical and

B₂ is --0--C(0)--NH--R₃ --NH-- wherein R₃ is an alkylene radical.

The chelating agent may be conjugated to the compound including ahydrophobic moiety by procedures known in the art. For example, thebis-anhydride of DTPA may be produced by refluxing pyridine and aceticanhydride. The anhydride may be conjugated to an amino group; forexample, a long chain di-alkylamine by a modification of the procedurereported by Takeshita (J.A.O.C.S. 59 #2 (Feb. 1982) wherein a solutionof the anhydride and triethylamine in tetrahydrofuran is reacted with along chain dialkylamine to produce an amphiphilic chelating agent.

A chelating agent may also be conjugated to cholesterol by use of aspacer compound. For example, diaminohexane and triethylamine dissolvedin isopropanol is reacted with a solution of beta cholesterolchloroformate, and the resulting product is conjugated to thebis-anhydride of DTPA by the procedure hereinabove described forconjugating the anhydride to a long chain alkylamine.

Procedures for producing amphiphilic chelating agent are deemed to bewithin the scope of those skilled in the art from the teachings herein.Procudures for conjugating chelating agents to other compounds are alsoapplicable to providing amphiphilic chelating agents in accordance withthe present invention.

The amphiphilic chelating agent forms a portion of the materials used inproducing the vesicle. The remaining materials used in forming thevesicle may be of a type known in the art, and include variousphospholipids, glycolipids, dialkylphosphates, lecithins, steroids, etc.Compounds which are suitable for producing vesicles are generally knownin the art and such compounds may be employed in conjunction with anamphiphilic chelating agent for producing vesicles in accordance withthe present invention.

In accordance with a preferred embodiment, a portion of the amphiphiliccompound(s) employed for producing the vesicle is derivatized with aligand whereby the vesicle wall includes a chelating agent and a ligand.In accordance with the preferred embodiment, a detectable metal markeris complexed to the chelating agent, whereby the vesicle may be employedas a tracer in an assay. In accordance with this aspect of theinvention, the tracer is comprised of a particle in which both thedetectable metal marker and the ligand form part of the particles;however, there is no covalent linkage between the ligand and detectablemetal. The detectable metal marker is complexed to an amphiphilicchelating agent, and another amphiphilic compound is derivatized with aligand to form separate portions of the vesicle wall; however, there isno covalent linkage between the detectable marker and the ligand.

The amphiphilic compound may be derivatized with a ligand by proceduresknown in the art. Such procedures may be similar to the proceduresemployed for conjugating a chelating agent to a compound including ahydrophobic moiety. Thus, for example, the ligand may be conjugated tothe amphiphilic compound through a spacer compound including tworeactive functional groups for conjugation to both the amphiphiliccompound and the ligand. Similarly, the ligand and/or the amphiphiliccompound may be derivatized so as to provide a functional group for suchconjugation. Similarly, the ligand and the amphiphilic compound mayinclude suitable functional groups for effecting the conjugation, suchas where the ligand includes an amino group and/or carboxyl group, andthe amphiphilic compound includes an amino and/or carboxyl group so asto provide for derivatization by an amide linkage. As known in the art,derivatization can be accomplished through a disulfide linkage. Sincesuch procedures are generally known in the art, no further details inthis respect are deemed necessary for a complete understanding of thepresent invention.

The vesicle wall is formed from a mixture of vesicle wall formingcomponents which include the amphiphilic chelating agent, and inaccordance with a preferred embodiment, an amphiphilic compoundderivatized with a ligand. The amount of amphiphilic chelating agentemployed in producing the vesicle is dependent upon both the amount ofthe detectable metal marker which is to be complexed to the vesicle, andthe ability to provide a vesicle with the desired properties. In mostcases, the amphiphilic chelating agent does not exceed 60 mole percentof the components employed in forming the vesicle, and generally doesnot exceed 50 mole percent. In general, the amphiphilic chelating agentis present in an amount of at least 5 mole percent. As hereinaboveindicated, higher and lower amounts may be employed, and the selectionof an optimal amount is deemed to be within the scope of those skilledin the art from teachings herein.

The amphiphilic compound derivatized with a ligand, when included informing the vesicle, is present in an amount dependent upon the ligand,and the assay in which it is to be employed. As known in the art,sensitivity is increased with lower amounts of ligand. The selection ofan optimum amount of ligand is deemed to be within the scope of thoseskilled in the art from the teachings herein.

Although it may be possible to form a portion of the wall of the vesicleso that it includes an amphiphilic chelating agent after the vesicle hasbeen formed (for example, by derivatizing cholesterol included as aportion of the wall of the vesicle with a chelating agent), theamphiphilic chelating agent is preferably provided prior to producingthe vesicle, and the vesicle is initially formed with such amphiphilicchelating agent. Similarly, although it is possible and known toderivatize an amphiphilic compound included in a formed sac with aligand, it is preferred to produce such an amphiphilic compoundderivatized with a ligand, prior to production of the vesicle.

Similarly, although it is possible to add a detectable metal forcomplexing with the amphiphilic chelating agent of the sac subsequent toformation of the sac, in accordance with a preferred embodiment, theamphiphilic chelating agent is complexed with the detectable metal,prior to production of the vesicle. In general, the metal is chelated tothe amphiphilic chelating agent to be employed in producing the sacunder conditions such that the chelating portion is ionized, with suchionization generally being accomplished at a pH of 6. After addition ofthe detectable metal, any detectable metal which is not chelated to theamphiphilic chelating agent, is removed; for example, by the use of gelfiltration.

The amphiphilic chelating agent, as well as other materials which are tobe used in producing the vesicle (including in accordance with apreferred embodiment an amphiphilic compound derivatized with a ligand)may be formed into a vesicle by procedures generally known in the art.For example, vesicles may be produced in accordance with the type ofprocedure generally described in U.S. Pat. No. 4,235,871. In accordancewith such a procedure, the vesicle wall forming components, includingthe amphiphilic chelating agent, and in accordance with a preferredembodiment, an amphiphilic compound derivatized with a ligand, in anorganic solvent are placed in a flask, and the solvent evaporated toform a film. Subsequently, an aqueous buffer, which contains the metalto be chelated or complexed to the sac, is added to form the sac,followed by sonication. Chelated metal is removed as hereinabovedescribed.

The above procedure and other procedures which are generally suitablefor producing vesicles are also suitable for the purposes of the presentinvention. In accordance with the present invention, the sac or vesiclemay be unilamellar or multilamellar. In most cases, a multilamellar sacis employed in that such a vesicle increases the amount of detectablemetal which can be incorporated into the sac.

The vesicles produced in accordance with the invention are highlymobile, and in general, the particles have a size in the range of from0.05 to 10.0 micron, in diameter. The vesicles, by use of the chelatingagent in appropriate amounts, are formed with a high ratio of detectablemetal atoms per vesicle, whereby there can be obtained an amplificationof signal in an assay. Moreover, the vesicles may also be provided witha plurality of ligand molecules, per vesicle.

In most cases, the vesicle includes a steroid component, as well as aglycolipid or phospholipid component, and such lipids may be provided ineither neutral form or in acid form.

The production of a suitable vesicle by proceeding in accordance withthe present invention should be apparent to those skilled in the artfrom the teachings herein in that the techniques which are generallyapplicable for producing vesicles are also applicable to producingvesicles including an amphiphilic chelating agent in accordance with thepresent invention.

Although the present invention has been hereinabove described withparticular reference to producing a vesicle which includes both adetectable metal marker and ligand wherein there is no covalent linkagebetween the marker and ligand, it is to be understood that includedwithin the spirit and scope of the present invention is the use of asolid particle other than a vesicle wherein the solid particle isderivatized with a chelating agent for complexing of a detectable metal,and is also derivatized with a ligand. In such a case, the solidparticle is provided with both ligand and detectable metal markerwithout covalent coupling of the ligand and the chelated metal markerfor use as a tracer in an assay. For example, solid particles may beproduced from styrene which is derivatized with an amino group; inparticular, polyamino-styrene, and the amino groups on the polystyreneparticles may be derivatized with chelating agent and ligand so as toprovide a solid particle having both detectable metal marker and ligandattached thereto for use as a tracer in an assay.

The detectable metals which may be chelated or complexed includeradioactive or non-radioactive metals. For example, radioactive metalsinclude radioactive cobalt. In addition, the detectable metal marker maybe formed from other than a radioactive metal, such as a materialpossessing the prcperties of paramagnetism, fluorescence orphosphorescence.

Rare earth metals of both the actinide and lanthanide series arepreferred. As representative examples, there may be mentioned terbium,prosium, europium, samarium, and neodimium.

The detectable metal marker is preferably a rare earth metal in thatsuch rare earth metal may be employed as a detectable marker in an assaywhich relies on fluorescence, and in particular, a time-delayfluorescent assay.

The ligand which is employed in conjunction with the solid particles,and in particular, in conjunction with a vesicle by derivatizing anamphiphilic compound employed in forming the vesicle, may be any one ofa wide variety of ligands for which there is a binding partner,sometimes referred to as an anti-ligand. The ligand, in general, is ahapten, antibody, or antigen.

In accordance with another aspect of the present invention, there isspecifically provided a hapten which is derivatized with a plurality ofdetectable metal atoms, with such detectable metal atoms preferablybeing a rare earth metal. The preferred rare earth metals are europiumor terbium, with europium being particularly preferred.

In accordance with this aspect of the present invention, for example, ahapten may be provided with a plurality of detectable metal atoms foruse as a tracer in an assay by forming a vesicle, which includes anamphiphilic chelating agent having a detectable metal chelated thereto,and an amphiphilic compound which is derivatized with a hapten.

As an alternative, such a hapten containing a plurality of detectablemetal atoms, for use as a tracer may be provided by derivatizing a solidparticle with both the hapten and a chelating agent, which chelates adetectable metal. The particle may be of the type hereinabove described.

Although proteins, and in particular, antibodies, have been derivatizedso as to provide such antibodies with a plurality of detectable metalatoms, such as a fluorescent rare earth metal, heretofore, it was notpossible to provide haptens with a plurality of detectable metal atoms,as a result of the low molecular weight of such haptens.

Thus, as should be apparent, in accordance with this one aspect of thepresent invention, there is provided a tracer, which is a hapten,wherein the hapten is provided with a plurality of detectable metalatoms, and specifically fluorescent rare earth metal atoms, and such atracer may be produced without the necessity of covalently coupling thedetectable metal marker to the hapten.

The vesicles which are formed in part from an amphiphilic chelatingagent having a detectable metal complexed thereto, and which inaccordance with a preferred embodiment, also include an amphiphiliccompound, which is derivatized with a ligand, can be provided as a driedmixture of the wall forming components, and such dried mixture can bereconstituted with water to provide a vesicle, which includes thedetectable metal marker. This is a departure from previous procedureswherein a sac or vesicle is provided with an encapsulated detectablemarker, which must be stored in water so as to permit effective use ofthe vesicle. In accordance with the present invention, the formedvesicle, may be dried, and then reconstituted and such reconstituted orreformed vesicle would include essentially all of the detectable metalmarker which was originally present in the sac, as well as the ligand.

Thus, for example, in accordance with this aspect of the invention, avesicle produced as hereinabove described may be dried, whereby the wallforming components, including the amphiphilic chelating agent having thedetectable marker complexed thereto may be stored in a dried form forsubsequent reforming into a vesicle by addition of water. The drying maybe readily accomplished by freeze drying; for example, cooling to -70°C. in dry ice acetone, followed by use of a freeze drying apparatus.

The freeze dried or dried vesicle may then be reformed by addition ofwater, such as a Tris-buffer at pH 8 to produce the vesicle, whichincludes the chelated detectable metal marker, as well as any ligandattached to an amphiphilic compound originally used in producing thesac.

As should be apparent, in this manner, it is no longer necessary tostore the vesicle in water, which increases the storage stability of thevesicle, and such vesicle may be effectively employed as a tracer in anassay by simply reconstituting the dried materials with water.

The vesicle, or any other particle, produced as hereinabove describedmay be employed as a tracer in an assay.

The ligand which is employed for derivatizing the sac is any ligand forwhich there is a corresponding binder and, in particular, is either anantibody, antigen or hapten, and the selection of the ligand isdependent upon the analyte to be determined. Thus, for example, if theassay is a competitive assay for determining an antigen or hapten, theligand employed in producing the tracer is either the analyte orappropriate analog thereof. The term "appropriate analog" means that theanalog of the analyte is bound by the binder for the analyte.

If the assay is a "sandwich" type of assay, then the ligand employed inproducing the tracer would be a ligand which is specific for the analyteto be assayed; for example, an antibody elicited in response to theantibody or antigen to be assayed. Alternatively, the antibody could bea monoclonal antibody.

The binder which is used in the assay is also dependent upon theanalyte. Thus, for example, if the analyte is an antigen or hapten, thebinder may be an antibody or a naturally occurring substance which isspecific for the analyte. If the analyte is an antibody, the binder maybe either an antibody, an antigen or naturally occurring substance whichis specific for the analyte.

The binder which is used in the assay may be employed in supported orunsupported form.

The tracer which is produced in accordance with the present inventionmay be determined in an assay, in most cases, without rupturing the sacor vesicle. More particularly, the chelating agent is generally ahydrophilic material, whereby such detectable marker is external to thebilayer which forms the sac or vesicle. It is to be understood, however,that when the term "external" is employed, with respect to the bilayerof the sac, it is meant that such external component may be inside oroutside of the sac and that the external portion of the bilayer is bothinside and outside of the sac. Thus, when the chelate is formed, aportion of the chelated metal is on the outside of the sac, rather thaninside of the sac and may be detected without rupturing of the sac. Itis to be understood, however, that the sac may also be ruptured in orderto determine the detectable metal. In most cases, it is preferred torupture the vesicle so as to insure that there is maximum amplification.

The overall procedure for the assay may be as generally practiced in theart with the determination of the detectable metal being accomplished bya procedure suitable for the particular metal employed.

In accordance with one assay procedure, the tracer formed in accordancewith the present invention may be employed for determining an analyte ina competitive type of assay wherein the tracer and analyte compete for alimited number of binding sites on a binder for the tracer and analyte.The presence and quantity of analyte in a sample may be determined bydetecting the tracer in the bound and/or free portions of the assay;i.e., tracer bound to binder, and/or tracer which does not bind to thebinder. As known in the art, the amount of tracer which becomes bound tothe binder is inversely proportional to the amount of analyte in thesample, and the quantity of analyte may be determined by comparing thevalue obtained in the assay with values obtained for the assay withknown quantities of analyte; i.e., a standard curve.

As hereinabove indicated, the detectable metal may be determined in somecases without rupturing of the sac, and in other cases the detectablemetal is determined by rupturing the sac. The sac may be ruptured byprocedures generally known in the art such as use of a detergent,enzymatic lysing, change in conditions of temperature, pH,concentration, etc.

The tracer may be included as a portion of a reagent kit or package foruse in an assay for an analyte. The tracer is included in a suitablepackage in a vial or container. The package may also include in suitablevials or containers a binder for the analyte, as hereinabove described.The binder may be provided in supported or unsupported form. The reagentkit or package may also include suitable standards (sample containingthe analyte in known concentrations), buffers, promoters, activatingcompounds, etc.

In accordance with a preferred procedure, the tracer is formed by use ofa vesicle to which a rare earth metal, such as europium, is chelated. Asknown in the art, europium may be detected by fluorescence.

The fluorescent rare earth metal, and in particular, europium, asgenerally known in the art, fluoresces when activated with a suitableactivating compound, such as beta-diketone or a dihydroxy compound, suchas sulfosalicylic acid.

The most widely used B-diketones are benzoylacetone (BA),dibenzoylmethane (DBM), thenoyltrifluoracetone (TTA),benzoyltrifluoroacetone (BTA), 1- and 2-naphthoyltrifluoroacetone(1-/2-NTA), acetylaceton (AcA), trifluoroacetylacetone (TFAcA), andhexafluoroacetylacetone (HFAcA). In addition to B-diketones the laseringproperties of different salicylate chelates have previously beeninvestigated and different methods for fluorometric determination oflanthanide ions (Eu, Tb, Sm, Dy) has been developed using thesecompounds and other ligands, such as terbium with dipicolinic acid (DPA)and with EDTA and sulpho-salicyl acid (SSA). Under favorable conditionsthe quantum yield of these chelates can be very high and come close to100 percent.

The beta-diketone may be provided as a chelate, which chelates the rareearth metal, which is also chelated by the amphiphilic chelating agentemployed in producing the vesicle. Use of a beta-diketone or otheractivating compound in this manner is disclosed in U.S. Pat. No.4,374,120, and such teachings are also applicable to the amphiphilicchelating agents, which are employed in producing a vesicle inaccordance with the present invention.

In accordance with a preferred embodiment, the rare earth metal iscaused to fluoresce by the use of a suitable activating compound, suchas a beta-diketone, after separating the rare earth metal from thechelate.

Thus, for example, a lanthanide, such as europium, may be separated fromthe amphiphilic chelating agent incorporated into the sac by use of asuitable detergent, such as Triton-X-100 at a low pH value. Afterseparation of the lanthanide, the fluorescence may be amplified by theuse of a suitable activating material, such as a beta-diketone. Inaddition, in order to improve the fluorescence, a Lewis base may beadded. Such bases are known, and are generally N-heterocyclic compoundssuch as o-phenanthroline, phosphines, and phosphine oxides.

Thus, in accordance with a preferred embodiment, the assay isaccomplished by employing a fluorescent rare earth metal as thedetectable marker, and fluorescence is read by procedures known in theart, with the preferred method being removal of the europium from thechelate, and activation thereof with a beta-diketone, and preferablyalso a Lewis base.

The assay may be preferably effected by a time delay fluorescent method;however, in some cases, it may be possible to determine fluorescencewithout a time delay, although a time delay is preferred in that iteliminates background fluorescence. The principles behind a time delayfluorescent assay are known in the art, and such principles are equallyapplicable to a time delay fluoresent assay in accordance with thepresent invention wherein the rare earth metal is chelated to a vesicleformed, in part, from an amphiphilic chelating agent, and in part froman amphiphilic compound derivatized with a suitable ligand.

Thus, for example, in such an assay, after separating bound and freecomponents, the europium is removed from the tracer, by use of adetergent, and by addition of a beta-diketone and a Lewis base. Theeuropium is then excited, for example, at 340 nm, and the beta-diketoneabsorbs energy and transfers the energy to the europium which fluorescesat 614 nm. Fluorescence may be determined in suitable instrumentationavailable in the art, after a suitable period for permitting decay ofbackground fluorescence, as generally practiced in the art.

Thus, for example, in a typical assay, a vesicle including anamphiphilic chelating agent having europium chelated thereto, and anamphiphilic compound derivatized with a suitable hapten is employed as atracer in a competitive assay wherein the binder is supported on a solidsupport such as a test tube. After incubation, the tubes are aspirated,whereby a tube includes only the bound tracer component. Subsequently, asolution containing a beta-diketone, a Lewis base, and a detergent, isadded to the tube. The detergent serves the dual purpose of lysing thevesicle, which is bound to the binder on the tube, and removing theeuropium from the chelating agent.

After a time period which permits decay of background fluorescence,fluorescence of the europium is determined on suitable instrumentationby excitation at 340 nm, and reading emission at 614. The amount oftracer bound to the tube is inversely proportional to the amount ofhapten in the sample, and the quantity may be determined by the use of asuitable standard curve prepared from standards having known amounts ofhapten.

Similarly, tracer in accordance with the present invention may beemployed in a "sandwich" type of assay, in which case, the tracer isformed from a vesicle including an amphiphilic chelating agent havingeuropium chelated thereto, and an amphiphilic compound derivatized withbinder for the analyte. The assay may be conducted in a tube coated witha binder for the analyte, and fluorescence determined as hereinabovedescribed. In this type of assay, the amount of tracer which is bound tothe tube through the analyte and coated binder is directly proportionalto the amount of analyte in the sample.

The assays which are practiced in accordance with the present inventionmay be employed in a wide variety of samples; in particular, body fluidssuch as serum, urine, sputum, etc. The selection of a suitable sample isdeemed to be within the scope of those skilled in the art from theteachings herein.

The assay of the present invention may be employed for determining awide variety of analytes. As representative examples of suitableanalytes, there may be mentioned viral antibodies, viral antigen,cardiac glycosides, such as digoxin and digitoxin, various drugs,including therapeutic drugs, and drugs of abuse, hormones, such as T₄,T₃ hCG, TSH, various steroids, and the like. These and other analytesshould be apparent to those skilled in the art from the teachings hereinand no further teachings in this respect are deemed necessary for a fullunderstanding of the invention.

The invention will be further described with respect to the followingexamples; however, the scope of the invention is not to be limitedthereby:

EXAMPLES A. Synthesis of Diethylenetriaminopentaacetic Acid--Dioctyldecylamine (DIPA-DODA)

The bis-anhydride of DTPA was prepared by refluxing DTPA (100 mmol, 38.5gm) in 120 ml of 1:1 v/v pyridine and acetic anhydride under a N₂atmosphere for 18 hours. The solid was filtered, washed with acetic acidanhydride and then washed with ether. The material was dried in a vacuumoven overnight at 125° C. and 70 microns. The yield was 95% (36 gm).

The typical synthetic procedure for (DTPA-DODA) was devised bymodification of Takeshita's protocol (JAOCS 59, No. 2, February, 1982,104-107). DTPA bis-anhydride (30 mmol, 11 gm) and triethylamine (20mmol, 2.02 gm) were suspended in 50 ml of tetrahydrofuran (THF) under aN₂ atmosphere and heated to 50° C. The DODA (5 mmol, 2.65 gm) wasdissolved in 10 ml of THF and added through an addition funnel over 1hr. The solution was stirred overnight. After being cooled, the mixturewas poured into a large excess of water (200 ml) and filtered. Theresidue was washed with water to give the crude product. The crudeproduct was dissolved in aqueous sodium hydroxide (120 ml) at pH 10. Thesolution was shaken with ether 2×(100ml). The ethereal and emulsifiedphases containing unreacted amine were discarded. The water phase wasneutralized with 10% HCl. The dissolved ether was removed by rotaryevaporation. The solution was adjusted to pH 4 with 10% HCl to yield aprecipitate. The precipitate was separated, washed with distilled waterand dried for 5 hours at 80° C. under vacuum. The dried material wasrecrystallized from ethanol and water. The material was characterized byTLC, IR, and NMR. The yield was 15% (600 mg).

B. Preparation of Digoxin-Labeled Europium-Bound Liposomes

Europium binding liposomes were prepared by placing 132 μmolecholesterol, 119 μmole distearoyl phosphatidylcholine, 13.2 μmoledistearoyl phosphatidylglycerol, 0.2 μmole distearoylphosphatidylethanolamine-digoxin conjugate and 62 μmolediethylenetriaminopentaacetic acid-dioctyldecylamino conjugate in 20 mlof chloroform-methanol (9:1 v/v). After evaporation of the organicsolvents, the remaining lipids were resuspended in 50 ml Tris buffer(Trimza Base 50 μmole, NaCl 117 μmole, pH 8.0) containing 31 μmoleEu(N03)3. This suspension was sonicated for 20 minutes at 100 watts (4C) and then centrifuged at 2,000 rpm for 5 min. The supernate was passedover a PD-10 gel filtration column and the liposome fraction collectedin the void volume. These liposomes were 200 nm in size as determinedwith the Coulter Particle Analyzer Model N4. The liposomes werelyophilized and reconstituted in the Tris buffer. The size distributionprofiles before and after lyophilization were very similar. Threeseparate batches of liposomes were prepared in this manner.

C. Assays Utilizing Digoxin-Labeled Europium-Bound Liposomes

A heterogeneous competition assay was developed using the previouslyprepared DTPA-DODA liposomes, anti-digoxin coated tubes and serumstandards of digoxin (BDI, Orangeburg). To the anti-digoxin coated tubeswere added 50 μl of digoxin serum standards (0-10 ng/ml), 50 μl ofliposomes, and 900 μl of Tris buffer. After incubation for 45 minutes at37° C., the tubes were aspirated and washed. Two ml of a solutioncontaining 10⁻⁵ M napthylenetrifluoroacetylacetonate, 10⁻⁵tri-N-octylphosphine oxide, 0.1% Triton X-100, 0.5% acetic acid, and 2%ethanol was added to each tube. The time-resolved fluorescence wasdetermined after 10 minutes utilizing a Spex L-111 instrument and thefollowing optimized parameters, 100 flashes per data point, excitation340 nm, emission at 614 nm, 0.5 ms delay, 0.25 ms window, and four slitsof 0/0/.5/.5 mm. Seven different concentrations of digoxin serumstandards were tested, and all samples were run in duplicate. Acalibration curve was constructed.

C. Synthesis of DiethylenetriaminopentaaceticAcid-Diaminohexane-Cholesterol (DTPA-DAH-Chol)

The typical synthetic procedure for (DAH-Chol) was devised bymodification of Rando's protocol (Biochimica et Biophysica Acta, 684(1982) 12-20). Diaminohexane (DAH), 26 mmol, 3.1 gm) and triethylamine(26 mmol, 2.67 gm) were dissolved in isopropanol (75 ml). With stirring,beta cholesterol chloformate (9 mmol, 4.05 gm) in benzene (9 ml), wasadded over 1 hr. After stirring overnight, the suspension was filteredand the filtrate taken to dryness. The solid precipitate was dissolvedin methylene chloride and extracted once with 10% Na₂ CO₂ and five timeswith saturated sodium chloride. The organic layer was dried over sodiumsulfate and taken to dryness. The solid was recyrstallized frommethanol. The material was characterized by TLC and IR. The yield was65% (3.6 gm).

The typical synthetic procedure for (DTPA-DAH-Chol) was devised bymodification of Takeshita's protocol (JAOCS 59 No. 2, February, 1982,104-107). DTPA bis-anhydride (29 mmol, 7.2 gm) and triethylamine (33mmol, 3.36 gm) were suspended in tetrahydrofuran (THF) (50 ml) under aN₂ atmosphere and heated to 50° C. The DAH-Chol (4 mmol, 2.3 gm) wasdissolved in THF (10 ml) and added through an addition funnel over 1 hr.The solution was stirred overnight. After being cooled, the mixture waspoured into a large excess of water and filtered. The residue was washedwith water to give the crude product. The crude product (2.5 gm) wasdissolved in CH₂ Cl₂ :MeOH:AcOH (20:4:0.1 v/v) and applied to a LS-2silica gel column equilibrated with the same solvent system. The productwas purified by flash chromatography. The product eluted between1,400-2,200 ml of solvent, and those fractions were pooled andevaporated. The material was characterized by TLC and IR. The yield was45% (1.75 gm).

E. Preparation of Digoxin-Labeled Europium-Bound Liposomes

Europium binding liposomes were prepared by placing 77 μmoleDTPA-DAH-Chol, 119 μmole distearoyl phosphatidylcholine, 13.2 μmoledistearoyl phosphatidylglycerol, and 0.2 μmole distearoylphosphatidylethanolamine-digoxin conjugate in 20 ml ofchloroform-methanol (9:1 v/v). After evaporation of the organicsolvents, the remaining lipids were resuspended in 50 ml Tris buffer(Trimza Base 50 μmole, NaCl 117 μmole, pH 8.0) containing 31 μmoleEu(NO₃)₃. This suspension was sonicated for 20 minutes at 100 watts (4°C.) and then centrifuged at 2,000 rpm for 5 min. The supernate waspassed over a PD-10 gel filtration column and the liposome fractioncollected in the void volume. These liposomes were 200 nm in size asdetermined with the Coulter Particle Analyzer Model N4. The liposomeswere lyophilized and reconstituted in the Tris buffer. The sizedistribution profiles before and after lyophilization were very similar.Three separate batches of liposomes were prepared in this manner.

F. Assays Utilizing Digoxin-Labeled Europium-Bound Liposomes

A heterogeneous competition assay was developed using the previouslyprepared DTPA-DAH-Chol liposomes, anti-digoxin coated tubes and serumstandards of digoxin (BDI, Orangeburg). To the anti-digoxin coated tubeswere added 50 μl of digoxin serum standards (0-10 ng/ml), 50 μl ofliposomes, and 900 μl of Tris buffer. After incubation for 45 minutes at37° C., the tubes were aspirated and washed. Two ml of a solutioncontaining 10⁻⁵ M napthylenetrifluoroacetylacetonate, 10⁻⁵tri-Noctylphosphine oxide, 0.1% Triton X-100, 0.5% acetic acid, and 2%ethanol was added to each tube. The time-resolved fluorescence wasdetermined after 10 minutes utilizing a Spex: L-111 instrument and thefollowing optimized parameters, 100 flashes per data point, excitation340 nm, emission 614 nm, 0.5 ms delay, 0.25 ms window and four slits of0/0/.5/.5 mm. Seven different concentrations of digoxin serum standardswere tested, and all samples were run in duplicate. A calibration curvewas constructed.

The present invention is particularly advantageous in that it ispossible to provide an amplified signal by incorporating a plurality ofdetectable metal atoms on a single particle. Moreover, it is possible toprovide a tracer and in particular, a hapten tracer, wherein there isprovided a detectable metal marker, which is not covalently linked tothe ligand portion of the tracer. Such a tracer is stable, and can bestored over long periods of time.

A further advantage of the present invention is that it is possible toprovide a time-delayed fluorescent assay, in which the signal isamplified, and in which the tracer remains stable. Moreover, such aresult can be achieved without the necessity of covalently linking theligand portion of the tracer to the fluorescent rare earth metal.

The invention offers the further advantage that it is possible toprovide amplification by the use of vesicles, without the storageproperties heretofore encountered when employing vesicles for suchpurposes.

These and other advantages should be apparent to those skilled in theart from the teachings herein.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, withing thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

What is claimed is:
 1. In an assay process for an analyte wherein atracer is detected in the assay, the improvement comprising:detecting inthe assay a tracer, said tracer comprising a vesicle derivatized with aligand, a portion of such vesicle wall being formed from an amphiphilicchelating agent having complexed therewith detectable metal atoms. 2.The process of claim 1 wherein the detectable metal atoms are comprisedof metal atoms which are fluorescent when complexed with an activatingagent.
 3. The process of claim 2 wherein the metal atoms are a rareearth metal.
 4. The process of claim 3 wherein the amphiphilic chelatingagent comprises from 5 to 60 mole percent of the materials forming thevesicle.
 5. The process of claim 3 wherein the ligand is a hapten. 6.The process of claim 3 wherein the ligand of the tracer is bound by theanalyte.
 7. The process of claim 3 wherein the ligand of the tracer isbound by a binder specific for the analyte.
 8. The process of claim 3wherein the amphiphilic chelating agent is a compound having thefollowing structural formula: ##STR4## wherein each R is an alkylenegroup or p-diazoniumphenyl substituted alkylene group wherein thealkylene group has from 2-4 carbon atoms and each R group may be thesame or different,X and Y are integers from 0-8, A is hydrogen or W; oneW group is ##STR5## or B--(PO₄)--(CH₂)₂ --NH--C(0)--CH₂ ⁻ ; or A₁ --B₂--C(0)--CH₂ -- wherein R' is an alkylene group having from 2-10 carbonatoms; R" is an aliphatic hydrocarbon having at least 11 carbon atomsand may be the same or different and Z is an integer of from 0-4, andthe remaining W groups are --CH₂ --C(0)--OR"' wherein R"' is hydrogen,an alkali metal or an amine, and B is ##STR6## wherein Y is an aliphatichydrocarbon radical having from 1-5 carbon atoms; each of B₁ isindividually --0--; --C(0)O--; or --CH₂ -- and may be the same ordifferent; and each R₁ is a substituted or unsubstituted hydrocarbonradical having at least 11 carbon atoms and may be the same ordifferent, and A₁ is cholesterol or cholestanol and B₂ is a radicalformed from a difunctional spacer compound.
 9. The process of claim 8wherein one of W is ##STR7## Y is 1, X and Z are zero, R is --CH₂ --CH₂--; and A is --CH₂ --C(0)--OR"'
 10. The process of claim 8 wherein oneof W is

    A.sub.1 --B.sub.2 --C(0)--CH.sub.2 --

wherein Al is a cholesterol radical and B₂ is

    --0 --C(0)--NH--R.sub.3 --NH-- wherein R.sub.3 is an alkylene radical.


11. The process of claim 3 wherein the rare earth metal is released fromthe vesicle, activated, and detected after a delay to permit decay ofbackground fluorescence.
 12. The process of claim 11 wherein the rareearth metal atom is activated by a beta-diketone and a Lewis base. 13.The process of claim 11 wherein the assay is a solid phase assay inwhich tracer is bound to analyte, an analyte is bound to a binderspecific for the analyte supported on a solid support.
 14. The processof claim 11 wherein the rare earth metal is europium.
 15. The process ofclaim 14 wherein the amphiphilic chelating agent comprises from 5 to 60mole percent of the materials forming the vesicle.
 16. The process ofclaim 3 wherein the rare earth metal is at least one member selectedfrom the group consisting of europium and terbium.
 17. The process ofclaim 16 wherein the analyte is in a serum sample and the tracerfraction is determined after a delay to permit decay of backgroundfluorescence.
 18. The process of claim 17 wherein the chelating portionof the amphiphilic chelating agent is comprised ofdiethylenetriaminepentaacetic acid.
 19. The process of claim 18 whereinthe hydrophobic portion of the amphiphilic chelating agent is comprisedof dialkyl amine.
 20. The process of claim 18 wherein the hydrophobicportion of the amphiphilic chelating agent is comprised of cholesterol.21. A reagent kit for use in an assay for an analyte, comprising:atracer comprising a vesicle derivatized with a ligand, a portion of saidvesicle wall being formed from an amphiphilic chelating agent havingcomplexed therewith detectable metal atoms.
 22. The kit of claim 21 andfurther comprising a binder specific for the analyte.
 23. The kit ofclaim 22 wherein the detectable metal atoms are comprised of rare earthmetal atoms which are fluorescent when complexed with an activatingagent.
 24. The kit of claim 23 wherein the rare earth metal is europium.25. The reagent kit of claim 23 wherein the ligand of the tracer isbound by analyte to be assayed.
 26. The kit of claim 23 wherein theligand of the tracer is bound by the binder specific for the analyte.27. The kit of claim 23 wherein the amphiphilic cheltating agentcomprises from 5 to 60 mole percent of the materials forming thevesicle.
 28. The reagent kit of claim 27 wherein the ligand of thetracer is a hapten.